To complete plasmid construction, we connected the skeleton (pUC57), promoter (pLldR), and target gene. We used two types of promoters: pLldR (referred to as plactate1) and pLldR-New (referred to as plactate2). The Lactate1 promoter is a lactate-responsive promoter that strongly initiates tumor drug expression in a lactate environment. This promoter is often used as a lactic acid biosensor for specific gene construction. Ana Zuniga's work shows that the plactate2 promoter is an optimized, synthetic lactate biosensor system designed to incorporate hybrid promoters of pLldR operators, remove glucose breakdown metabolites and hypoxia inhibition, and adjust the expression of regulatory genes and reporter genes. Plactate2 can still activate the expression of tumor drugs in the low oxygen environment3. Therefore, we attached plactate1 and plactate2, respectively, to three different target genes (sfGFP, Azurin, and Anti-PDL1) to screen for the optimal lactic acid promoter pLldR and transferred the constructed recombinant plasmid to E. coli Nissle1917. The follow-up will provide a new idea for tumor treatment.

The target gene sfGFP was obtained by PCR. sfGFP is a fluorescent protein, and the addition of sfGFP allows us to detect the success of plasmid construction. At the same time, the ability of the two promoters to initiate expression can be further compared. We obtained promoter (plactate1 and plactate2) and sfGFP fragment of the target gene by PCR amplification. Then, we detected the DNA length by gel electrophoresis and obtained that the length of plactate1 homologous arm-sfGFP and plactate2 homologous arm-sfGFP were both 750bp (as shown in Figure 1).

Figure 1. The identification of PCR production by agarose gel electrophoresis
Left: the graph shows plactate 1 and plactate 2 has the length of 3150bp and 3800bp.
Right: the graph shows that p1-sfGFP and p2-sfGFP have a length of 750bp.

Then, through homologous recombination, we connected the target gene sfGFP and promoter (plactate1 and plactate2) to the skeleton to obtain Plactate1-SFGFP, Plactate2-sfGFP and then verified the successful construction of the recombinant plasmid by colony PCR. As can be seen from Figure 2-A, the amplified band was 750bp, which was consistent with the expected result, indicating that the initial construction of the recombinant plasmid was successful. Subsequently, we extracted the constructed plasmid and sent it to a biological company for sequencing. The sequencing results were compared with the sfGFP sequence one by one. As shown in Figure 2-C, the gene sequences of the two plasmons were matched entirely, which further proved that the recombinant plasmid was successfully constructed.

Figure 2. PCR identification of plactate1-sfGFP plasmid.
A. The graph shows that the lengths of p1-sfGFP and p1-sfGFP are both 750 bp. B. The graph shows the flora growing in petri dish. C. The graph shows the sequencing measured by the bio company.

According to Ana Zuniga's article, Azurin, a cuproredoxin isolated from Pseudomonas aeruginosa, can enter cancer cells and induce apoptosis. It interacts with specific molecules on the surface of cancer cells to disrupt intracellular signaling pathways. The pLldR promoter, derived from Lactobacillus plantarum, is a lactate-induced promoter known for its ability to respond to lactic acid. It is a moderately strong promoter that controls the expression level of target genes. We attached Azurin and Anti-PD-L1 to the skeleton (pUC57) with plactate1 and plactate2, respectively. We obtained promoters (plactate1 and plactate2) and target genes (Azurin and Anti-PD-L1) by PCR amplification. Next, we measured the length of the PCR products by gel electrophoresis. The lengths of plactate1 homologous arm-Azurin, plactate2 homologous arm-Azurin, plactate1 homologous arm-Anti-PD-L1, and plactate2 homologous arm-Anti-PD-L1 are all 750bp (as shown in Figure 3-A).

Figure 3 The identification of PCR production of plasmid and Azurin/Anti-PD-L1gene. A. PCR production by agarose gel electrophoresis
B. The proof diagram of PCR

Then, through homologous recombination, we attach target genes (Azurin and Anti-PD-L1) to the skeleton, plactate1-Azurin, plactate2-Azurin, plactate1-Anti-PD-L1, and plactate2-Anti-PD-L1 were obtained, and then colony PCR was used to verify whether the recombinant plasmid was successfully constructed. As can be seen from Figure 4-A, the amplified bands were 750bp, which was consistent with the expected result, indicating that the initial construction of the recombinant plasmid was successful. Subsequently, we extracted the constructed plasmid and sent it to a biological company for sequencing. The sequencing results were compared with the sequences of the target genes (Azurin and Anti-PD-L1) one by one. As shown in Figure 4-C, the gene sequences of the two plasmids were matched entirely, which further proved that the recombinant plasmid was successfully constructed.

Figure 4 PCR identification of the recombinant plasmid plactate1-Azurin, plactate2-Azurin plactate1-Anti-PD-L1, and plactate2-Anti-PD-L1 A. The PCR production identification by agarose gel electrophoresis.
B. The graph shows the flora growing in petri dish.
C. The graph shows the sequencing measured by the bio company.

We used alkaline lysis to extract plasmids (plactate1-Azurin, plactate1-Anti-PD-L1, plactate2-Azurin, plactate2-Anti-PD-L1) from bacterial cultures. Next, we converted the recombinant plasmid to EcN1917 competent by heat shock transformation. PCR was used to verify whether the plasmid was transformed into EcN1917. The results are shown in Figure 5-A. The colony PCR results were detected by agarose gel electrophoresis, and the amplified band was 550bp, which was consistent with the target band, indicating that the four plasmids were successfully transformed into EcN1917.

Figure 5 PCR identification of EcN1917 transformants
A. The PCR production of the plactate1-Azurin, plactate2-Azurin, plactate1-Anti-PD-L1, and plactate2-Anti-PD-L1 transformants by agarose gel electrophoresis.
B. The graph shows the flora growing in petri dish.

We formulated different lactic acid concentrations (0mM, 2mM, 5mM, 10mM) to simulate different tumor microenvironments. The optimal promoter was selected by comparing the fluorescence protein intensity and the expression concentration of tumor drugs, which provided a new idea for future tumor treatment.

The constructed strains (plactate1-sfGFP, plactate2-sfGFP) were expanded in culture, and different concentrations of lactic acid (0mM,2mM,5mM,10mM) were used to induce the expression of GFP fluorescent protein, as shown in Figure 6. It can be seen from Figure 6 that when the lactate concentration is 5mM, the fluorescence protein expression of the two strains is the strongest. Through horizontal comparison, the expression of the fluorescent protein of Plactate2-GFP is more vital than that of Plactate1-GFP, and it can be concluded that the plactate2 promoter has a stronger activation expression ability than the plactate1 promoter.

Figure 6 The effects of lactic acid concentration on the expression of fluorescence protein

In order to further determine the optimal conditions for the growth of bacterial solution, we set up different bacterial solution cultures of OD600 (OD0.3, OD0.6, OD0.8, and OD1.0) and different lactate induction concentrations (0mM, 2mM, 5mM, and 10mM). Four strains were cultured to OD0.3, OD0.6, OD0.8, and OD1.0, and different concentrations of lactic acid were added for induction. After induction, the two target proteins were extracted by ultrasonic crushing, and the concentration of target proteins was detected. As shown in Figure 7, when OD600 is 0.6 and lactic acid induced concentration is 5mM, the protein expression level is the highest. Therefore, the optimal conditions for protein expression can be determined as OD600=0.6 and lactic acid concentration is 5mM.

Figure 7 The effects of bacterial concentration and lactic acid concentration on the expression of recombinant proteins

Further, we processed the data using Origin Pro to form a three-dimensional mapping of the expressed protein concentrations of the four EcN1917 strains under different conditions (Fig. 8). By analyzing the 3D mapping, we can conclude that as far as the culture OD600 of the bacteriophage is concerned, there is a significant difference in the concentration of target proteins when induction is performed at different bacteriophage OD600. As can be seen from Figure 8, the highest level of protein expression was observed when the OD600 was 0.6. As for the lactic acid induced concentration, it is not the case that the higher the induced concentration, the highest the concentration of the target protein. As can be seen from Figure 8, when the final concentration of lactic acid was added to induce the expression, the highest level of the target protein was expressed.

Figure 8 3D mapping of the expressed protein concentrations

We further examined the change in the pH value of bacterial solution after different concentrations of lactic acid induced culture. The bacterial solution cultured overnight at 37 degrees Celsius was tested with a pH meter. When the lactic acid concentration is 0mM, the pH value of the bacterial solution is 6.87. When the lactic acid concentration is 2mM, the pH value of the bacterial solution is 6.60. When the lactic acid concentration is 5mM, the pH value of the bacterial solution is 6.42. When the lactic acid concentration is 10mM, the pH value of the bacterial solution is 6.08. When a tumor develops, the pH of the tumor microenvironment is about 6.5 to 7.2. When the pH value is less than 6.5, it will inhibit the killing effect of immune cells, thereby increasing the survival rate of tumor cells. When the pH value exceeds 7.2, it will affect the metabolism of tumor cells, membrane permeability, and other physiological processes, thus inhibiting its growth and spread. Therefore, when probiotics infiltrate tumors in a high lactic acid and low pH environment, tumor drugs are released to achieve the purpose of killing cancer cells.

Table 1 pH value of bacterial solution after different concentrations of lactic acid

After the optimal protein expression conditions were obtained, the constructed strains (plactate1-Azurin/Anti-PD-L1, plactate2-Azurin/Anti-PD-L1) were expanded for culture, and 5mM lactic acid was used to induce the actual expression level of tumor drugs in EcN1917. The experimental results are shown in Figure 8. The size of the Azurin protein is about 19kDa, and the Anti-PD-L1 protein is about 24kDa, consistent with the expected results. A large amount of target protein indicates that the Azurin/Anti-PD-L1 protein is successfully expressed. In the future, we will further study the expression and tumor inhibition effect of the constructed probiotics in the tumor environment.

Figure 9 Detection of Azurin and Anti-PD-L1 protein expression by SDS-PAGE